Project Summary During cancer progression, angiogenesis is upregulated to supply the ever-increasing metabolic demands of the growing tumor. While targeting tumor-associated angiogenesis has been a therapeutic strategy for many years, these techniques demonstrate limited effectiveness in many cancer types. We believe this may be due to limited understanding of the biomechanical environment of the tumor. Recently cancer-associated fibroblasts (CAFs) have been shown to be key regulators of the peritumoral environment responsible for secreting several growth factors that control angiogenesis and metastasis. CAFs exhibit a myofibroblast-like phenotype, with increases in alpha-smooth muscle actin and Snail1. We hypothesized that CAF-generated increases in biomechanical strains enhance tip cell activation and drive angiogenesis in the tumor microenvironment. Our initial work has demonstrated that CAF biomechanical activity is directly related to the vascularization potential of these cells in in vitro models of vascular growth, and that inhibiting the mechanotransductive pathways in these cells abrogated their ability to support the formation of blood vessel networks. Continuing this research will further elucidate the roles of biomechanics during tumor progression as well as reveal potential targets for novel anti-cancer therapeutic strategies. During the K99 portion of the grant, we will (1) investigate the role of CAF biomechanics in an in vivo angiogenic mouse model and (2) optimize a microfluidic platform for angiogenesis studies that will allow for isolation and interrogation of biomechanical parameters. The proposed microtissue platform will be highly innovative in that it allows for independent control of several key biomechanical properties. Importantly, this phase of the grant will complement the PI?s career development by incorporating training in cancer cell biology analysis techniques as well as mouse models of cancer progression. During the R00 portion of the grant, we will investigate how endothelial cells respond to mechanical cues from CAFs utilizing the microtissue model previously developed. Finally we will investigate potential anti-cancer therapeutic strategies targeting CAF biomechanical promotion of tumor development in a mouse model of breast cancer. Ultimately this work has significant implications for not only understanding biomechanics of cancer progression but also the development of a unique in vitro microtissue model that will permit interrogation of biomechanics in a truly original manner. The training, techniques, and approaches developed during this grant should open several new avenues for future studies and will allow the PI to transition into a fully-independent investigator.